Electroprocessed collagen

ABSTRACT

A matrix of collagen is obtained through the method of electroprocessing. As a common natural polymer, collagen may be electroprocessed to form a matrix for multiple different applications.. The flexibility and variability of the processing allows the collagen matrix to be predesigned to meet many applications. These applications are included, but not limited to, biomedical applications, manufactured leather applications, food casing products, and footwear and clothing products.

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 09/512,081, filed Feb. 24, 2000, which is acontinuation-in-part of U.S. patent application Ser. No. 09/386,273,filed Aug. 31, 1999. The '081 application also claims priority, in part,from U.S. Provisional Application Serial No. 60/121,628, filed on Feb.25, 1999.

[0002] This invention relates to the product of electroprocessingcollagen. Because collagen is a commonly-found natural polymer, thereare many varied uses for a matrix of electroprocessed collagen fibersand/or droplets.

BACKGROUND OF THE INVENTION

[0003] Many different types of synthetic polymers are used to formstructures or platforms in biomedical applications. Bandages and patchesare formed of woven polymer fibers. Prosthetics can be made of differentpolymers and combinations of polymers. Vascular sleeves, for instancecan be made of woven fibers. Several problems with using man-madepolymers for biomedical applications include difficulty in mimickingnatural tissue and likelihood of rejection by a body's natural defensemechanisms.

[0004] In order to overcome problems with using synthetic polymers, anatural polymer such as collagen can be used. Collagen is conventionallyused in biomedical applications as a gel or foam. (E.g., U.S. Pat. No.5,891,588). There are limitations to this use of collagen as a result ofthe inherently unoriented nature of a collagen gel or foam. In order toobtain orientation and more closely mimic biological conditions,collagen is typically processed, if at all, by extrusion techniques.These techniques are limited by diameter or thickness of fibers orsheets available from the equipment used.

[0005] In another field, a common, naturally-occurring product thatincorporates collagen is leather. Artifical leather products made of,for instance, vinyl are known, but they are not as desirable as naturalleather itself. Leather is a well known product that has been used forthousands of years and for a large range of applications. Leather is amaterial for making different clothing and fashion accessory productssuch as coats, gloves and purses, among other things. Leather is used inthe manufacture of shoes and boots. Leather is used to form differentupholstery products in the home and, for instance, in automobiles.Leather is also used as an industrial product with numerousapplications.

[0006] There are limitations inherent in the use of leather as a rawmaterial. Leather has naturally occurring imperfections, which meansthat it is difficult to obtain a consistent product in consistentsupply. It is necessary to adapt any specific application for leatherproduct to available leather products instead of adapting the leather tothe application. For instance, seams are needed in the formation ofcomplex shaped leather goods. Also, it is virtually impossible to repairtears or holes in leather products.

[0007] In addition to biomedical platforms and leather, collagen is acommon food casing. For instance, meat products can include an outsidecasing that incorporates collagen. A well-known example is sausage wherea casing of animal intestine (collagen) is filled with seasonings, meatand other ingredients. Drawbacks with conventional meat casingtechnologies include a limited range of diameters of casings that areavailable and limited consistency with the quality of those casings.

[0008] In still another separate field, the textile industry has usedfor many years the process of electrospinning polymer fibers to makefabrics and nonwoven products. The polymers employed are typicallypolyester and other synthetic polymers. Traditionally, sheets ofelectrospun polymer fabric are formed and then processed (cut and sewn,for instance) just like any other sheets of woven or knit fabric.

SUMMARY OF THE INVENTION

[0009] Accordingly, it is an object of the present invention to overcomethe foregoing limitations and drawbacks by using collagen as the polymerin the process of electroprocessing. By electroprocessing collagen, amatrix can be formed for use in multiple fields of use includingbiomedical applications, food casing applications, manufactured leatherapplications, and footwear and clothing applications, among others.

[0010] In one embodiment, the invention is the product of the process ofelectroprocessing collagen. This processing may comprise electrospinningcollagen fibers, or it may comprise electrospraying collagen droplets.Further, the collagen may be synthetically manufactured collagen orcollagen produced by genetic engineering or a subset of a collagenmolecule such as a specific sequence of amino acids contained within alarger collagen protein molecule.

[0011] A method for making a matrix of collagen includes providing asubstrate and a reservoir of solution including collagen wherein thereservoir has an orifice that allows the solution to leave thereservoir. Then, either the substrate or the solution is electricallycharged and the other is grounded. The collagen is then streamed fromthe reservoir and through the orifice onto the substrate to form amatrix. The step of streaming collagen may form a matrix of collagenfibers. It may, alternatively, form a matrix of collagen droplets.Further, the substrate may define a preselected shape. The collagenmatrix can be formed in the presence of cross-linking agents or may betreated with a cross-linking agents after streaming.

[0012] A further method for making a matrix of collagen includesproviding a substrate, a target, and a reservoir solution includingcollagen wherein the reservoir has an orifice that allows the solutionto leave the reservoir. Then, either the target or the solution iselectrically charged and the other is grounded. The substrate isdisposed between the orifice and the target. The collagen is thenstreamed from the reservoir and through the orifice onto the substrateto form a matrix. The step of streaming collagen may form a matrix ofcollagen fibers. It may, alternatively, form a matrix of collagendroplets. Further, the substrate may define a preselected shape. Thecollagen matrix can be formed in the presence of cross linking agents ormay be treated with cross linking agents after streaming.

[0013] In still a further embodiment, there is a food casing comprisinga matrix of electroprocessed collagen. The collagen may be electrospuncollagen fibers. Alternatively, it may be electrosprayed collagendroplets. The collagen matrix may be cross linked or otherwise processedto alter its structural or chemical properties.

[0014] In a still further embodiment, manufactured leather comprises amatrix of electroprocessed collagen. The collagen may includeelectrospun collagen fibers. Alternatively, the matrix may includeelectrosprayed collagen droplets. Also, the collagen may be cross linkedor otherwise processed to alter its structural or chemical properties.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIGS. 1 and 2 are scanning electron micrographs of differentmagnification of the matrix of electroprocessed collagen as described inExample 1.

[0016]FIG. 3 is a transmission electron micrograph of the matrix ofelectroprocessed collagen as described in Example 1.

[0017]FIGS. 4 and 5 are scanning electron micrographs of differentmagnification of a matrix of electroprocessed collagen/elastin asdescribed in Example 2.

[0018]FIG. 6 is a scanning electron micrograph of the matrix ofelectroprocessed collagen and elastin as described in Example 3.

[0019]FIGS. 7 through 9 are scanning electron micrographs of varyingmagnification of electroprocessed collagen to form leather as describedin Example 4.

[0020]FIGS. 10 through 12 are scanning electron micrographs of a matrixof collagen that has been electroprocessed and treated with a crosslinking agent to form leather as described in Example 4.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0021] This description includes different examples of electroprocessingtechniques. There are other examples contained in U.S. patentapplication Ser. Nos. 09/512,081, 09/386,273 and U.S. ProvisionalApplication Serial No. 60/121,628. Those applications are incorporatedherein by reference as if set forth in their entirety.

[0022] The term “electroprocessing” shall be used broadly to cover themethods of electrospinning of fibers, electrospraying(electroaerosoling) of droplets, combinations of electrospinning andelectroaerosoling, and any other method where a polymer includingcollagen is streamed across an electric field. The solution beingstreamed may be charged and directed to a grounded substrate. Similarly,the solution may be streamed from a grounded reservoir in the directionof a charged substrate. The term “electroprocessing”, therefore, is notlimited to the specific examples set forth herein.

[0023] Throughout this application, the term “solution” is used todescribe the liquid in the reservoir of the electroprocessing method.This could imply that the collagen polymer is fully dissolved in theliquid. In this application, the term “solution” also refers tosuspensions when the collagen is not soluble (or only partially soluble)in the liquid used in a given process. This broad definition isappropriate in view of the large number of solvents or other liquidsthat may be used in the many variations of electroprocessing.

[0024] Also, the term “collagen” is used throughout in its broadestdefinition. There are multiple types of collagen that arenaturally-occurring as well as types that are being syntheticallymanufactured or produced by genetic engineering. Other types may befound or synthesized in the future, or specific subsets of variouscollagen molecules may be isolated with unique effects and may be usedto make products like those described herein. All of these types andsubsets are encompassed in the use of the term “collagen” herein. Also,as evidenced by some of the examples, the collagen may be mixed withother polymers during electroprocessing to obtain specifically desirableproperties for given end use of the matrix.

[0025] There are many different applications for electroprocessedcollagen. The versatility is enabled by the variability of the processitself. Generally speaking, there is variability with the equipmentused, the solution that is streamed in the process, and variouspost-process treatments.

[0026] In the most fundamental sense, the electroprocessing apparatusincludes a streaming mechanism and a target substrate. The streamingmechanism will include a reservoir or reservoirs to hold the solutionthat is to be streamed in the process. The reservoir or reservoirs haveat least one orifice or nozzle to allow the streaming of the solutionfrom the reservoirs. There may be a single nozzle or there may bemultiple nozzles in a given electroprocessing apparatus. If there aremultiple nozzles, they may be attached to one or more reservoirscontaining the same or different solutions. Similarly, there may be asingle nozzle that is connected to multiple reservoirs containing thesame or different solutions. Also, the size of the nozzle may be variedto provide for increased or decreased flow of the solution out of thereservoir through the nozzle. A pump used in connection with thereservoir may be used to control the flow of solution streaming from thereservoir through the nozzle or nozzles. The pump may be programmed toincrease or decrease the flow at different points during anelectroprocessing run.

[0027] The target substrate may also be used as a variable feature inthe electroprocessing of polymers such as collagen. Specifically, thetarget may be the actual substrate onto which the polymers includingcollagen are deposited. Alternatively, a substrate may be disposedbetween the target and the nozzle. For instance, a petri dish can bedisposed between a nozzle and a target, and a matrix can be formed inthe dish to study cell growth in 3-D by having a scaffold in the bottomof the dish. Other variations include non-stick surfaces between thenozzle and target. The target may also be specifically charged(grounded) along a preselected pattern so that the polymer streamed fromthe orifice is directed into specific directions. Ideally, the electricfield is controlled by a program to create a matrix having a desiredgeometry. The target and the nozzle or nozzles may be engineered to bemovable with respect to each other thereby allowing additional controlover the geometry of the matrix to be formed. It is envisioned that theentire process will be controlled by a microprocessor that is programmedwith the specific parameters to obtain a specific, preselectedelectroprocessed matrix of collagen and, if desired, other polymers.

[0028] Also, as noted in the specific examples that follow, the nozzleor orifice that allows streaming of solution from the reservoir is shownto be charged and the target is shown to be grounded. Those of skill inthe electroprocessing arts will recognize that the nozzle and solutionmay be grounded and the target may be electrically charged. In anyevent, it is the creation of the electric field and the effect of theelectric field on the streamed collagen that helps create the uniquecollagen matrix.

[0029] In addition to the multiple equipments and variations andmodifications that can be made to obtain desired results, similarly thesolution can be varied to obtain different results. For instance, thesolvent or liquid in which the collagen is dissolved or suspended may bevaried. The collagen can be mixed with other polymers to obtaineddesired end results. In still a further variation, when multiplereservoirs are used, the ingredients in those reservoirs may beelectrosprayed separately or joined at the nozzle so that theingredients in the various reservoirs may react with each othersimultaneously with the streaming of the solution into the electricfield. Also, when multiple reservoirs are used, the differentingredients in different reservoirs may be phased in over time in theprocessing period. The collagen may be directly altered, for example, byaltering the carbohydrate profile of the molecule. Also, other materialsmay be attached to the collagen before, during or afterelectroprocessing. Further, the temperature and other physicalproperties of the process can be modified to obtain different results.

[0030] Finally, there are many types of post-process treatments that maybe used to modify and adjust the matrix that is the result of theelectroprocessing procedure. For instance, a matrix of electroprocessedcollagen may be treated with a cross linking agent, including chemicaland UV-light based cross-linking agents. Also, the matrix may be treatedwith variations in temperature. Still further chemical variations may beenvisioned by those desiring specific end properties of a matrix.

[0031] Having considered some of the variations in the process that areavailable, it is clear that there are multiple different applicationsthat would benefit from the electroprocessing of collagen. The differentfields of use of this process and the product thereof include biomedicalapplications, food casing applications, manufactured leatherapplications, and clothing and footwear applications. Of course, theseare not the only applications possible for an electroprocessed matrix ofcollagen. Examples of potential applications in these fields of use willbe discussed in the following.

[0032] Biomedical Applications

[0033] There are at least two major biomedical applications forelectroprocessed collagen matrices. In general terms, there is use of amatrix in the formation of an extracellular matrix and in the formationof a drug delivery platform.

[0034] Specific applications and variations where electroprocessedcollagen is used as a drug delivery platform are described inprovisional application entitled entitled Electroprocessing In DrugDelivery And Cell Encapsulation, Ser. No. ______, filed Oct. 18, 2000,which is incorporated herein by reference as if set for in its entirety.

[0035] Some of the variations and potential applications for use of anelectroprocessed matrix of collagen as an extracellular matrix arediscussed in detail in prior filed pending applications, Ser. Nos.09/386,273, 09/512,081 and 60/121,628. Those applications areincorporated herein by reference as if set forth in their entirety.

[0036] Electrospun collagen has a repeating, banded pattern when it isexamined by electron microscopy. This banded pattern is typical ofcollagen fibrils produced by natural processes (i.e. banded pattern isobserved in collagen when it is produced by cells). Most collagenfibrils produced in vitro lack this banded appearance. This bandedpattern is an important attribute because it allows cells to have accessto active sites within the collagen molecule that promote or regulatespecific activities. For example, the AP-15 site, a 15 amino acidsequence within the collagen molecule, promotes bone cells (for exampleosetoblasts) to produce and secrete hydroxyapatite, a critical componentof bone. In many of the types of collagen fibrils that are produced invitro, the AP-15 site is cryptic and hidden. When osteoblasts are placedonto a collagen matrix with a cryptic AP-15 site, they fail to expressbone specific components. By modulating the electrospinning system,different binding sites can be selectively hidden or exposed to controlcell behavior. In addition, collagen may be enriched with specificsequences during, after or before the electrospinning process.Furthermore, specific subsets of collagen polymers, such as the AP-15site can be produced and selectively electrospun so that the entirematrix is composed of just polymers of the AP-15 binding sites. Otherspecific sites and sequences within the collagen molecules may bemanipulated and processed in a similar fashion—for example the RGDbinding sites of the integrin molecule. Increasing the amount of RGDbinding sites within a matrix might be used to increase the efficiencyor strength of attachment of cells to a collagen matrix.

[0037] In addition to the extracellular matrix (tissue scaffold)applications described in the earlier-filed applications, the followingare further applications for use of electroprocessed collagen as anextracellular matrix.

[0038] 1. Vascular Valves—Natural material or collagen derived fromgenetic engineering or an entirely synthetic source, with or withoutcells could be used in this application. A valve, for example a cardiac,aortic, or venus valve, could be assembled in vitro most likely with abioreactor for leaflet preconditioning. The advantage of utilizingelectroprocessing is that it affords one the ability to mimic thearchitecture of the collagen in a native leaflet. Many variations couldbe accounted for in the production of such a valve. One variation is adesign where a ring is formed around the edge of the valve that isthickened like the natural valve. This ring provides a means ofattachment and structural support.

[0039] 2. Tendon/Muscle Repair—A matrix is formed in the shape of asleeve that is hollow and shaped like a funnel. At the base of thefunnel, the solid tendon will project. The sleeve part can fully orpartially envelop the muscle belly. This would allow a firm attachmentor a not so firm attachment that could be gradually loaded as the musclecells grow into the new tendon. This method could be utilized in vitrobut also in vivo for tissue repair/regeneration. Increasing the numberof RGD binding sites within an electrospun matrix of collagen used in atendon might be used to increase the adhesion of muscle cells to thematrix.

[0040] 3. Tendon or Ligament—An entire ligament can be spun with theslight twist created by such means as a rotating nozzle or air vortex.The collagen can be autologous in nature (this holds true for the allpossible utilizations of electrospun collagen). One possibility forcreating a ligament would be to harvest the damaged ligament, grind itup in a crude mixture and then electroprocess the new ligament. Thiswould take place on the hours time scale so that one may harvest oneday, spin, and reimplant the next day. It might be possible toelectroprocess on sight in situ.

[0041] 4. Vascular Scaffold or Support—An additional product is not anartery but an aneurysmal sleeve. This is done by making a collagensleeve with or without additional reinforcement such as a suture oranother thick fiber. One could spin a tube, cut the aneurysm (in mostcases one may not wish to cut the aneurysm but simply wrap it or wrapone which has torn), and then slip the vascular support over thetransected/torn artery. Once in place, the surgeon could then stitch thesupport directly to the arterial wall. The sleeve covers the aneurysmsight and acts like a sock surrounding it to reinforce its structure.

[0042] 5. Wound Care—A dressing can be applied topically. This could bea simple bandage to a complex wound packing for a diabetic type ulcer orbed sore. Again, note that drug impregnation (antibiotics, hormones,angiogenic factors etc.) during or after electrospinning of the fibrousmats is a strong component for wound care products. Gene delivery isanother possible application by incorporating vectors or naked geneticmaterial in the spinning process to be incorporated by the cellularcomponents during consumption and reorganization of the electrospunmatrix. Also, one could incorporate oligoneculotides into the matrix.Antisense and sense oligoneculotides or even full length gene sequencescould be added to provide transient control of gene expression. As thematrix is altered, the oligonucleotides would be released and becomeavailable to the cells. Another possible addition would be proteaseinhibitors. Also, one could incorporate peptides that physically repelor attract cells depending on a desired response. Materials such aschondroitin sulfate can be added to the matrix during after or beforeelectrospinning to modify its structure. Chondroitin sulfate is used tostabilize collagen against heat denaturation and is a naturalcross-linking agent.

[0043] 6. Cartilage—Again, this could be autologous. In this case,hyaluronic acid, either natural or synthetic versions that are or arenot subject to breakdown can be added to the collagen prior, during orafter electroprocessing to promote hydration of the engineered tissue.Proteoglycons can be added to more closely mimic cartilage. One couldalso add cells in vitro and/or in vivo depending on final designs.

[0044] 7. Hernia Patch—Basically this is the same concept as theaneurysm sleeve except to repair hernias. Instead of a sleeve, thehernia patch would consist of a sheet of electrospun collagen.Additional natural materials such as elastin or synthetic materialscould be added prior to, during or after electrospinining. This productwould replace the Teflon patches currently used.

[0045] 8. Nerve Repair—Seamless tubes of collagen, with or withoutbiologically active molecules such as nerve growth factor or othermaterials such as various drugs can be used to facilitate repair ofdamaged or severed nerves.

[0046] 9. Bone—Collagen mats or tubes can be seeded with osteoblastswith and without biologically active molecules such as bone morphogenicprotein to generate bone of various shapes and sizes used, for example,for maxillofacial repair. As noted specific subsets of collagenmolecules such as the AP-15 site can be used to alter the biologicalactivity of an electrospun matrix. A matrix composed entirely of theAP-15 sequence could be electrospun or a matrix partially enriched withthe AP-15 site or other sites could be fabricated.

[0047] 10. Suture Material—Threads or cables of collagen could beproduced by electrospinning for use as sutures. An electrospun suturebased on collagen would be biocompatible as well as bioresorable. Aswith other materials fabricated from collagen the material and orchemical properties of the electrospun suture could be controlled prior,during, or after the fabrication process. Notably the suture materialcould be designed as a drug delivery system to suppress inflammation,promote healing, etc. Scaling the suture to larger sizes, i.e. producinga thicker thread rather than a fine thread, would allow filaments to beused in other applications. For example, given sufficient thickness orstrength, a synthetic catgut for stringing tennis rackets could befabricated from collagen.

[0048] 11. Bioengineering Platform—An additional application of theinvention is as a platform for the fabrication of tissue using anelectrospun matrix as a solid support. The fabrication platform iscomposed of an electrospun matrix of collagen, or other microfibers orblends of material. The overall three-dimensional geometric shape of theplatform is determined by the ultimate design and type of tissue to bebioengineered.

[0049] Several permutations of the design are possible. For example, tofabricate a solid three-dimensional “plug” of tissue, a fabricationplatform is electrospun on a mandrel. The mandrel may be cylindrical inshape, a flattened oval shape, a rectangular envelope shape (like amailing envelope) or any other desired shape. The bioengineeringplatform was electrospun on a mandrel with the desired shape and allowedto “dry”. The electrospun matrix was removed from the mandrel. For acyclindrical-shaped bioengineering platform or any other shape ofconstruct in which an enclosed area is desired, a suture, glue, stapleor heat seal or some other method may be used to seal one end of thebioengineering platform. This results in a hollow platform that isclosed on one end and open on the other. The electrospun platform cannow be filled with cells or other materials, or cells or other materialsmay be placed on the outer surface of the construct. For example, amixture of collagen or other materials and cardiac or skeletal musclecells or any other desired cell type may be placed within the platform.The free and open end of the envelope that was used to fill theconstruct with material can be sutured, glued or heat sealed shut toproduce an enclosed bioengineering platform. The entire construct isthen placed into a bioreactor for cell culture or directly placed insitu for further development. With modifications, a bioengineeringplatform composed of a solid, rather than a hollow, format can beelectrospun.

[0050] As before specific subsets of the collagen molecules or fiberblends may be used to form the matrix or supplement the material,physical, structural or chemical properties of the bioengineeringplatform. For example, the AP-15 site sequence might be added to asolution of collagen during electroprocessing, attached to collagenprior to processing, after electrospinning, streamed from a separatesource, or even streamed alone to form a matrix.

[0051] Variations to this tissue engineering process includes thefollowing:

[0052] Add endothelial cells to the core or outside surface of thebioengineering platform during seeding to pre-form a capillary network.

[0053] Form a cylinder and seed the outer surface with smooth musclecells to form an arterial construct. The inner surface can then beseeded separately with endothelial cells to form an endothelial lining.

[0054] Mix/add cells during the electroprocessing process to directlytrap or surround cells within the matrix as it is in the process offorming. This may be accomplished by adding cells from a separatenozzel(s) or other sources if the materials to be electroprocessed mustbe placed in an organic solvent or solvents that will not support lifefor some other reason (for example a solvent with high or low pH or asolvent with high or low salt content).

[0055] Cell polarity can be regulated by controlling the orientation ofthe fibers on the mandrel during or after electrospinning. For alignedfibers, this can be accomplished by electrospinning onto a targetmandrel that is spinning. The fibers will be wrapped around the mandrelin the direction of rotation. Electrospinning onto a static,non-spinning mandrel will produce a more random fibrillar matrix.

[0056] Polarity may also be controlled by first electroprocessing analigned matrix onto a spinning mandrel. The matrix is then cut from themandrel, rotated 90 degrees (or any other degree of rotation) and placedback onto a mandrel. A second layer of material is then electrospun ontothe first layer. This method will produce an inner layer of fibers thatare aligned along the long axis of rotation.

[0057] Cell polarity can be regulated by placing the bioengineeringplatform within a stretching device installed in the bioreactor. Bygradually applying strain across the construct over time the cellswithin the platform will spread in parallel with the applied force.

[0058] Mix cells in suspension with the fabrication platform as it iswithin the bioreactor to coat the exterior and or interior surfaces. Forexample, add tendon fibroblasts to the exterior of a skeletal muscle“plug” to establish the exterior architecture.

[0059] Use the electrospun bioengineering platform as a differentiationplatform for the manipulation of stem cells. The porosity and chemicalcomposition of the electrospun matrix can be controlled prior, during,and after fabrication. This will allow an investigator to create thetype of microenvironment that is believed to be critical for controllingthe differentiation process in stem cells.

[0060] Electrospin conductive materials into the matrix. In this way theconstruct can be electrically stimulated to promote neural ingrowth orcontraction of engineered muscle. Incorporating conductive materialsinto an electrospun matrix also could be used as means to further alterthe properties of a matrix following fabrication. For example, applyingan electrical field across an existing matrix might be used to alter theshape, porosity or density of the matrix. The stability of the matrix(resistance to breakdown) might be altered—either increased ordecreased, by applying an electric field across the filaments.

[0061] Electrospin magnetically active materials into the matrix. Inthis way, the construct could be induced to alter shape or position byapplying a magnetic field across the bioengineering platform. Forexample, applying a magnetic field across an existing matrix might beused to alter the shape, porosity or density of the matrix. Thestability of the matrix (resistance to breakdown) might bealtered—either increased or decreased by applying a magnetic fieldacross the filaments.

[0062] Place the engineered tissue within the omentum forvascularization.

[0063] Place the engineered tissue or electrospun matrix directly insitu.

[0064] There are advantages to this tissue engineering process. Itprovides a solid support that can be used to grow tissue at very highdensity. It controls and establishes a local microenvironment with theconstruct. By controlling material properties of the matrix, one cancontrol the buoyant nature of the construct, the porosity and thestability of the matrix (i.e. it can be designed to be very stable or todegrade over a relatively short period of time). It provides a platformfor cell culture or tissue bioengineering that is unique and amenablefor use in a bioreactor environment. And it provides a platform for cellculture and bioengineering of many different types of tissue that islarge and can be manipulated manually.

EXAMPLE 1

[0065] The collagen used was Type I (calf skin, Sigma Chemical Co.). Thecollagen was suspended in 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) at aconcentration of 0.1181 grams in 3 ml HFP. Once in solution orsuspension (solution a milky color), the solution was loaded into a 1 mlsyringe plunger. A 15-gauge luer stub adapter was then placed on thesyringe to act as the electrospinning nozzle and charging point for thecontained collagen solution. The filled syringe was placed in the KDScientific's syringe pump set to dispense the solution at rate of 18ml/hr utilizing a Becton Dickinson 1.0-ml syringe plunger. The positivelead from the high voltage supply was attached to the luer stub adaptermetal portion. The syringe pump was turned on and the high voltagesupply turned on and set at 20 kV. The grounded target was a 303stainless steel mandrel (0.6 cm W×0.05 cm H×4 cm L) placed approximately6 inches from the tip of the adapter. The mandrel was rotated atapproximately 500 rpm during the spinning process. In the experiment, 1ml of the collagen solution was electrospun to form a nice, white mat onthe grounded mandrel. After electrospinning, the collagen mat wasremoved from the mandrel and processed for scanning electron microscopyevaluation. The results of this fibrous mat production can be seen inFIGS. 1 and 2. (Magnification 1000× and 4300× respectively). The matproduced was approximately 200 microns thick.

[0066] Transmission electron microscopy (TEM) evaluation was done and isshown in FIG. 3. This cross-sectional micrograph illustrates theapproximate 100 nm collagen fiber diameter and the typical 64 nm bandingindicative of native collagen polymerization.

EXAMPLE 2

[0067] The methods for this example are the same as Example 1 except forthe electrospun solution. In this case, the spinning solution consistedof 0.1155 grams collagen, 0.1234 grams of elastin from ligamentum nuchae(Fluka), and 5 ml HFP. In this experiment, 2 ml of the suspension wasspun to form the mat. The results of this experiment are shown in FIGS.4 and 5. (Magnification 1800 and 6500 respectively). The mat producedwas approximately 50 microns thick.

[0068] In this example, elastin was incorporated. For additionalvariations, other polymers, peptides, growth factors, biochemicals maybe added during or after the spinning process to the collagen matsproduced.

EXAMPLE 3

[0069] The methods for this example are the same as Example 1 except forthe electrospun solution. In this example, the solution electrospun wascomposed of 0.067 grams of type I collagen, 0.067 grams of type IIIcollagen, 0.017 grams of elastin from ligamentum nuchae, and 2 ml HFP(44% Type I, 44% Type III, and 12% elastin). This is a ratio similar tothat found in native arterial wall tissue. An example of the results ofthe collagen-based fibrous mat production can be seen in FIG. 6(magnification 1800×). The mats produced were approximately 100 micronsthick.

[0070] Food Casing Applications

[0071] A matrix of electroprocessed collagen may be used as a foodcasing product. In one example, a mandrel having a specific shape(typically in the case of a sausage—a cylindrical shape) is used as thetarget for the streamed solution containing collagen. The thickness andstrength of the matrix can be varied depending on the requirements ofthe food product to be packed within the matrix. Specifically desirablevariations in this process may include the incorporation in the solutionof various flavor or food preservative ingredients that will beincorporated into the food casing matrix. Alternatively, flavoring orpreservatives may be added to the matrix after electroprocessing. Thecolor of the matrix can also be varied. Aside from simple cylindricalshapes, more complex shapes can be seamlessly made. Trendy food productshaving unusual shapes are possible.

[0072] Manufactured Leather

[0073] Since leather is comprised essentially of collagen, theelectroprocessing invention may be used to manufacture a leatherproduct. Complex, seamless leather forms can be fabricated. Thisfabrication process would utilize the natural polymer collagen infibrillar form to produce natural leather like products. Electrospinningleather in this fashion will provide a means to make fiber blends toproduce novel fabric combinations. Novel fabric combinations could bemanufactured to exhibit unique physical properties such as increasedelasticity, water resistance/water proofness, increased strength,durability, and selected incorporation of resilient materials forpadding and gripping. Additionally, the thickness of the leather fabriccan be selected and controlled. This fabrication process furtherminimizes waste during production of the electrospun leather as well asfinding a use for waste natural leather created as a by product ofworking with natural leather products. Also, utilizing natural productsis more environmentally safe.

[0074] Still further, the product and process provides a mechanism tomend natural leather or the electrospun leather described herein. Theinvention therefore minimizes waste resulting from imperfection or tearsin natural leather. Additionally, electrospun leather could be combinedwith natural leather to produce hybrids that would minimize waste bycapitalizing on the utilization of scrap materials typically discardedin current production methods.

[0075] Seamless materials are more waterproof and less likely to failthan standard seamed leather products. Also, it is possible to producecomplex seamless three-dimensional shapes with electroprocessing thatprecisely fit complex shapes. Another advantage is that all of theleather would be of premium quality. The invention eliminates the needto discard a rawhide because of tears or other imperfections, becausethe invention fabricates the electrospun leather from the basic collagenfibers that make up leather. The quality of the manufactured leatherwould be absolutely uniform and dependent upon the selection and choicesin the manufacturing process, not the limitations in the raw materials.

[0076] Collagen from rawhide, or any other source can be isolated andprepared for electroprocessing. For example, hide can be cut into smallpieces, frozen and fragmented into small pieces, lyophilized and used asa crude mixture of raw material for electroprocessing. Other isolationprocedures are also possible, i.e., acid hydrolysis or the isolation ofcollagen to form a gel dispersion. These procedures can be tailored toisolate collagen in a realtively pure form or a crude form (i.e., stillmixed with the components elements that make up the hide in its rawstate). Acid extracts of collagen may be dialized against other solventsfor example water, to prepare the collagen for processing.

[0077] The collagen or crude extract can then be suspended in1,1,1,3,3,3-hexafluoro-2-propanol or another appropriate solvent orsuspension. The solution (or suspension) is then placed into a syringeor other source, charged to high voltage and directed at a groundedtarget. Streams of solvent containing the suspended collagen aredirected at the target. As the stream bridges the gap between the sourceand ground, the collagen undergoes polymerization to form filaments.

[0078] As with any electrospinning process, the filament diameter andorientation can be regulated to a high degree by the reactionconditions. Adding other specific materials into the collagen canfurther modify material properties. For example, adding a naturalmaterial like elastin or a synthetic material like rubber can beexpected to produce leather with novel elastic properties. Materialproperties can also be modulated by adding additional materials duringthe electrospinning process from additional sources (i.e. othersyringes). The advantage of this strategy is that filaments ofdissimilar properties can be mixed at the molecular level duringfabrication, i.e. filaments of separate and distinct properties can beintermingled at the individual filament level. Spinning specificmaterials in sequence with one another can produce layers of materials.Also, by mixing different types of collagen or collagen that has beenmanipulated other ways (e.g. added or removed carbohydrates or peptides)in the solution or filament form, different textures or materialproperties can be achieved. Also, by forming a gradient in the collagensources, the composition of the final product can be controlled. Acollagen gradient would allow the materials to take on multiplemechanical properties within the same panel of fabric to allow thefabric to accomplish complex functions. The gradient may includevariable concentrations of collagen and/or variable types of collagen orother polymer or additive. For example, a high concentration of collagencould be used to produce filaments of high mechanical strength. Agradient towards a low concentration of collagen in the source solutionwould produce less filamentation and more globular material that couldprovide a soft surface, a gripping surface, or padding.

[0079] After electrospinning, further processing can be performed toproduce varying colors, textures, scents, and resiliency (i.e. tanning).Cross linking agents (for example gluteraldehyde, UV light or otherconventional tanning materials) can be applied to the product at variousstages to adjust material properties. Also, there is nothing to prohibitusing the final product in more traditional ways, e.g., producing sheetsof the manufactured leather fabric to make products with seams.

EXAMPLE 4

[0080] The collagen used was Type I (calf skin, Sigma Chemical Co.). Thecollagen was suspended in 1,1,1,3,3,3-hexfluoro-2-propanol (HFP) at aconcentration of 0.1181 grams in 3 ml HFP. Once in solution orsuspension (solution in milky color), the solution was loaded into a 1ml syringe plunger. A 15-gauge luer stub adapted was then placed on thesyringe to act as the electrospinning nozzle and charging point for thecontained collagen solution. The filled syringe was placed in the KDScientific's syringe pump set to dispense the solution at a rate of 18ml/hr utilizing a Becton Dickinson 1.0-ml syringe plunger. The positivelead from the high voltage supply was attached to the luer stub adaptermetal portion. The syringe pump was turned on and the high voltagesupply turned on and set at 20 kV. The grounded target was a 303stainless steel mandrel (0.6 cm W×0.05 cm H×4 cm L) placed approximately6 inches from the tip of the adapter. The mandrel was rotated atapproximately 500 rpm during the spinning process. In the experiment, 1ml of the collagen solution was electrospun to form a nice, white mat onthe grounded mandrel. After electrospinning, the collagen mat wasremoved from the mandrel and processed for scanning electron microscopyevaluation. The results of this fibrous mat production can be seen inFIGS. 7-9. (Magnification 800×, 8000× and 850× respectively). The matproduced was approximately 200 microns thick.

[0081] For the production of leather, the collagen mat sample was placedin 2% glutaraldehyde solution (0.1 M sodium cacodylate) for three days(over the weekend). The sample was then placed in 1% osmium tetroxidefor 1 to 1.5 hours. The sample was then dehydrated with increasing ethylalcohol solutions (50-100%). The samples were then sputter coated forviewing on the scanning electron microscope. Results of the fixed sampleare shown in FIGS. 10-12. (Magnification 800×, 8000× and 2700×respectively). The figures show a highly cross-linked collagenous matthat is a reproduction of leather.

[0082] Footwear and Clothing Applications

[0083] The use of electroprocessing to manufacture footwear and clothingproducts is examined in more detail in provisional application entitledElectroprocessing Polymers To Form Footwear And Clothing, Ser. No.______ filed on Oct. 18, 2000 which is incorporated herein by referenceas is set forth in its entirety. In short, a substrate having a desiredshape, whether it be a shoe, sock, shirt, or any article of clothing isthe target in the electroprocessing procedure. In this way, a customshoe or custom piece of clothing is made that will exactly andseamlessly fit the shape of the madrel—ideally the shape of a person'sfoot, torso, etc. The thickness and attributes of the resulting matrixmay be varied depending on the type of clothing that is desired. Also,different polymers may be incorporated into the solution to obtain theend result of a specifically desired piece of clothing or footwear.

[0084] While the invention has been described with reference to specificembodiments thereof, it will understood that numerous variations,modifications and additional embodiments are possible, and accordingly,all such variations, modifications, and embodiments are to be regardedas being within the spirit and scope of the invention.

What is claimed is:
 1. The product of the process of electroprocessingcollagen.
 2. The product described in claim 1, wherein the process ofelectroprocessing comprises electrospinning collagen fibers.
 3. Theproduct described in claim 1, wherein the process of electroprocessingcomprises electrospraying collagen droplets.
 4. The product described inclaim 1, wherein the collagen comprises synthetically manufacturedcollagen.
 5. A method for making a matrix of collagen comprising:providing a substrate, providing a reservoir of solution comprisingcollagen wherein the reservoir has an orifice that allows the solutionto leave the reservoir, electrically charging either the substrate orthe solution, and grounding the other of the substrate or the solutionthat is not electrically charged, and streaming the collagen onto thesubstrate to form a matrix.
 6. The method described in claim 5, whereinthe step of streaming the collagen onto the substrate forms a matrix ofcollagen fibers.
 7. The method described in claim 5, wherein the step ofstreaming the collagen onto the substrate forms a matrix of collagendroplets.
 8. The method described in claim 5 wherein the substratedefines a preselected shape.
 9. The method described in claim 5, furthercomprising treating the collagen matrix with a cross-linking agent. 10.The method described in claim 5, wherein the collagen comprisessynthetically manufactured collagen.
 11. The method described in claim5, wherein the collagen comprises a subset of a collagen molecule.
 12. Amethod for making a matrix of collagen comprising: providing asubstrate, providing a target, providing a reservoir of solutioncomprising collagen wherein the reservoir has an orifice that allows thesolution to leave the reservoir, electrically charging either the targetor the solution, and grounding the other of the target or solution thatis not electrically charged, disposing the substrate between the orificeand the target, and streaming the collagen onto the substrate to form amatrix.
 13. The method described in claim 12, wherein the step ofstreaming the collagen onto the substrate forms a matrix of collagenfibers.
 14. The method described in claim 12, wherein the step ofstreaming the collagen onto the substrate forms a matrix of collagendroplets.
 15. The method described in claim 12, wherein the substratedefines a preselected shape.
 16. The method described in claim 12,further comprising treating the collagen matrix with a cross-linkingagent.
 17. The method described in claim 12, wherein the collagencomprises synthetically manufactured collagen.
 18. The method describedin claim 12, wherein the collagen comprises a subset of a collagenmolecule.
 19. A food casing comprising a matrix of electroprocessedcollagen.
 20. The food casing described in claim 19, wherein thecollagen comprises electrospun collagen fibers.
 21. The food casingdescribed in claim 19, wherein the collagen comprises electrosprayedcollagen droplets.
 22. The food casing described in claim 19, whereinthe collagen is cross-linked.
 23. A method of making a food casingcomprising electroprocessing a matrix of collagen.
 24. Manufacturedleather comprising a matrix of electroprocessed collagen.
 25. Themanufactured leather described in claim 24, wherein the collagencomprises electrospun collagen fibers.
 26. The manufactured leatherdescribed in claim 24, wherein the collagen comprises electrosprayedcollagen droplets.
 27. The manufactured leather described in claim 24,wherein the collagen is cross-linked.
 28. A method of manufacturingleather comprising electroprocessing a matrix of collagen.
 29. Themethod described in claim 28, further comprising the step of treatingthe collagen matrix with a cross-linking agent.